2.3.5. Lactic Acid Bacteria

Lactic acid bacteria (LAB) as facultative anaerobic bacteria are part of the original microflora of mackerel tuna flesh; hence, the number of these bacteria can increase under both aerobic and anaerobic conditions [59]. As shown in Figure 6F, it is clear that the initial number of LAB was 1.59 log<sup>10</sup> CFU/g and did not exceed 4.88 log<sup>10</sup> CFU/g in control fillets until the 15th day of the storage period. It was also observed that the LAB counts of the XAN and XAN-EEP fillet samples were significantly lower (*p* < 0.05) compared to the control samples (uncoated fillets) during the refrigerated storage period.

The best treatment in terms of inhibiting LAB proliferation in mackerel tuna fillet samples among the other tested groups was XAN-EEP 2% compared to the other tested groups, and this could be attributed to the synergistic antimicrobial effect of EEP. It has been known that LAB bacteria are the most resistant Gram-positive bacteria to antimicrobial agents [60]. The results of our study confirmed this as LAB bacteria were more resistant compared with other spoilage bacteria versus XAN combined with EEP.

This conclusion regarding the synergistic antimicrobial effect of EEP may agree with what was indicated by Duman and Özpolat [61] concerning the effect of aqueous extract of propolis during storage of shibuta (*Barbus grypus*) fillets at 4 ◦C. It was observed that 0.5% aqueous extract of propolis significantly reduced the number of shibuta's lactic acid bacteria at all storage times compared to the control samples. Duman and Özpolat [61] attributed this effect to the phenolic content of propolis.

In another study conducted in Greece and Cyprus on evaluating the antibacterial activities of propolis ethanolic extracts (PEs), the results concluded that the minimum inhibitory concentration (MIC) of all studied propolis ethanolic extracts was higher for lactic acid bacteria compared to the other tested bacterial species (*Listeria monocytogenes*, *Staphylococcus aureus*, and *Bacillus cereus*) [62].

#### 2.3.6. Yeasts/Molds 2.3.6. Yeasts/Molds

The yeast and mold species are common agents of microbial spoilage in refrigerated fish [63]. A in prior investigations, the primary count (day 0) of yeast/mold of mackerel tuna fillets was 2.13–2.16 log<sup>10</sup> CFU/g (Figure 6G) [44,64]. It was shown from the results of all treatments (XAN-EEP 0%, XAN-EEP 1%, and XAN-EEP 2%) in the present study that they had a significant ability (*p* < 0.05) to reduce the number of yeasts/molds compared to the untreated fillet samples (control) under cooling conditions (Figure 6G). These results were in agreement with Duman and Özpolat [61], confirming the antifungal activity of propolis from refrigerated shibuta fillets. The yeast and mold species are common agents of microbial spoilage in refrigerated fish [63]. A in prior investigations, the primary count (day 0) of yeast/mold of mackerel tuna fillets was 2.13–2.16 log10 CFU/g (Figure 6G) [44,64]. It was shown from the results of all treatments (XAN-EEP 0%, XAN-EEP 1%, and XAN-EEP 2%) in the present study that they had a significant ability (p < 0.05) to reduce the number of yeasts/molds compared to the untreated fillet samples (control) under cooling conditions (Figure 6G). These results were in agreement with Duman and Özpolat [61], confirming the antifungal activity of propolis from refrigerated shibuta fillets. Other studies have highlighted the role of propolis extracts against Candida tropicalis

Other studies have highlighted the role of propolis extracts against *Candida tropicalis* and *Candida albicans* [62], and also against *Aspergillus niger* and *Candida albicans* [57]. and Candida albicans [62], and also against Aspergillus niger and Candida albicans [57].

#### *2.4. Sensory Evaluation* 2.4. Sensory Evaluation

It is worth noting that the use of xanthan and ethanolic extract of propolis as food-grade components in coating-forming makes it safe for consumers [65,66]. The freshness of mackerel tuna fillets during storage was assessed sensorially by taste, odor, and overall acceptability. At the beginning of the storage period, all groups of fillets were characterized by a smell of fresh fish and a distinctive shiny surface, but with the continuation of the cold storage process, the sensory properties of the samples deteriorated, but the rate of deterioration was significantly faster (*p* < 0.05) in the uncoated fillet samples (control) compared to the samples coated with xanthan/ethanolic extract of propolis (Figure 7A–C; Supplementary Table S1). According to Bazargani-Gilani and Pajohi-Alamoti [67], the permissible sensory level must be higher than 4 for the samples of fish fillets to be fit for consumption. Fishy and putrid odors increased gradually in control after 10 days of storage. Microbial damage and the consequent accumulation of receptors, such as trimethylamine (TMA) and biogenic amines, are the cause of unpleasant odors [68]. The results obtained in our study from the panelists in terms of the general acceptance of all the fillet samples under examination showed that: (a) uncoated fillet samples (control) had a shelf life of fewer than 11 days; (b) treatment with XAN-EEP 0% had a viability of more than 11 days; (c) treatment with XAN-EEP 1%, viable for 15 days; (d) treatment with XAN-EEP 2% had a viability of 20 days. It is worth noting that the use of xanthan and ethanolic extract of propolis as foodgrade components in coating-forming makes it safe for consumers [65,66]. The freshness of mackerel tuna fillets during storage was assessed sensorially by taste, odor, and overall acceptability. At the beginning of the storage period, all groups of fillets were characterized by a smell of fresh fish and a distinctive shiny surface, but with the continuation of the cold storage process, the sensory properties of the samples deteriorated, but the rate of deterioration was significantly faster (p < 0.05) in the uncoated fillet samples (control) compared to the samples coated with xanthan/ethanolic extract of propolis (Figure 7A–C; Supplementary Table S1). According to Bazargani-Gilani and Pajohi-Alamoti [67], the permissible sensory level must be higher than 4 for the samples of fish fillets to be fit for consumption. Fishy and putrid odors increased gradually in control after 10 days of storage. Microbial damage and the consequent accumulation of receptors, such as trimethylamine (TMA) and biogenic amines, are the cause of unpleasant odors [68]. The results obtained in our study from the panelists in terms of the general acceptance of all the fillet samples under examination showed that: (a) uncoated fillet samples (control) had a shelf life of fewer than 11 days; (b) treatment with XAN-EEP 0% had a viability of more than 11 days; (c) treatment with XAN-EEP 1%, viable for 15 days; d) treatment with XAN-EEP 2% had a viability of 20 days.

**Figure 7.** *Cont*.

(B)

Figure 7. The response surface plot of coating treatments on the (A) taste, (B) odor, and (C) overall acceptability of mackerel tuna fillet samples during storage at 2 °C for 20 days. 0.0 \*: Control samples [Uncoated mackerel tuna fillet samples (soaked samples in sterile distilled water)]. 0.00 \*\*: XAN-EEP 0% [Coated samples with xanthan containing (0%) ethanolic extract of propolis]. 1.00: XAN-EEP 1% [Coated samples with xanthan containing (1%) ethanolic extract of propolis]. 2.00: XAN-EEP 2% [Coated samples with xanthan containing (2%) ethanolic extract of propolis]. These results can be attributed to the fact that the incorporation of EEP into the XAN **Figure 7.** The response surface plot of coating treatments on the (**A**) taste, (**B**) odor, and (**C**) overall acceptability of mackerel tuna fillet samples during storage at 2 ◦C for 20 days. 0.0 \*: Control samples [Uncoated mackerel tuna fillet samples (soaked samples in sterile distilled water)]. 0.00 \*\*: XAN-EEP 0% [Coated samples with xanthan containing (0%) ethanolic extract of propolis]. 1.00: XAN-EEP 1% [Coated samples with xanthan containing (1%) ethanolic extract of propolis]. 2.00: XAN-EEP 2% [Coated samples with xanthan containing (2%) ethanolic extract of propolis].

coating significantly (p < 0.05) preserved the general acceptability scores and the fresh These results can be attributed to the fact that the incorporation of EEP into the XAN coating significantly (*p* < 0.05) preserved the general acceptability scores and the fresh organoleptic characteristics of taste and aroma in trout meat until the last period of time. These results are also in agreement with what was reported by Duman and Özpolat [61] about shibuta fillets.

The results of general acceptance of the studied treatments can be linked to the antimicrobial effect of EEP associated with its content of phenolic compounds [47].
